An important subset of human breast cancer is characterized by over- expression of the epidermal growth factor receptor (EGFR). This sub-group constitutes about 30% of all breast cancers, and over-expression of EGFR has been repeatedly shown to be an indicator of poor prognosis. The overall goal of this grant is to use a novel expression cloning strategy, in conjunction with a unique panel of breast cancer and normal breast epithelial cell lines, to determine the causal molecular alterations that induce specific altered growth phenotypes pf EGFR-positive human breast cancer cells. The specific aims of this proposal are: 1.) To functionally clone and identify genes from retroviral expression libraries derived from EGFR over-expressing breast cancer cells that induce EGFR- independent proliferation of normal and immortalized human mammary epithelial cells. 2.) To functionally clone and identify genes from retroviral expression libraries derived from EGFR over-expressing breast cancer cells that induce anchorage independent growth capacity in a panel of human mammary epithelial cells with varying abilities to survive in soft agar. 3.) To use differential display PCR, and high density oligonucleotide arrays, to analyze changes in gene expression in growth factor independent or anchorage independent clones isolated in these experiments. High titer retroviral expression libraries will be developed, using HBC cell lines developed in our lab, that over-express EGFR without gene amplification. These cells do not have amplifications of the common breast oncogenes, but do have amplified regions of their genome as determined by comparative genomic hybridization. Retroviral expression libraries will be transduced into a panel of well characterized human mammary epithelial cells, and transformants will be selected on the basis growth factor or anchorage independent growth. Inserts will be rescued by PCR using primers that bind to vector sequences, and then sequenced. This approach will allow us to identify and functionally clone breast cancer oncogenes directly from well characterized human breast cancer cells.